We have been studying the effects of black widow spider venom and nerve stimulation on the ultrastructure, electrophysiological properties and acetylcholine contents of neuromuscular junctions. We have purified from the venom a protein that abolishes virtually all of the electrophysiological activity of nerve terminals and depletes them of their vesicles. The protien has a molecular weight of about 130,000 and has no lypolytic or proteolytic activity. We plan to study the effects of temperature, Ca ions and pH on the activity of the protein in an attempt to determine its mechanism of action. We have also found that venom-treated neuromuscular junctions retain most of their acetylcholine even though they have lost most of their vesicles and exhibit virtually no electrophysiological activity. We plan to study the release of acetylcholine from these vesicle-free preparations. These results suggest that the amount of acetylcholine contained in the vesicles is only a small fraction of the total store. This in turn suggests that studies of the turnover of acetylcholine in vesicles may best be accomplished by electrophysiological means. Therefore we plan to measure the changes in the amplitude and frequency of miniature end-plate potentials during stimulation to exhaustion in the absence of drugs. BIBLIOGRAPHIC REFERENCES: Ceccarelli, B. and Hurlbut, W.P. 1975. The effects of prolonged repetitive stimulation in hemicholinium on the frog neuromuscular junction. J. Physiol. 247, 163-188. Ceccarelli, B. and Hurlbut, W.P. 1975. Transmitter release and the vesicle hypothesis. in Golgi Centennial Symposium Proceedings. M. Santini, ed. Raven Press, N.Y. pp. 529-545.